Rroutine Investigation Of Vibrio Parahaemolycus And Detection Of 3 Kinds Of Vibrio Parahaemolycus By Loop Mediated Isothermal Amplification (LAMP)

Vibrio parahaemolyticus is an important foodborne pathogen. When people eat foods contaminated by it, diarrhea, vomiting and other symptoms,even dehydration, shock and death will occur. Currently, the scale of food poisoning caused by Vibrio parahaemolyticus shows a tendency to expand and it is the primary food-borne pathogen on China. Therefore, it is particularly important to carry out routine investigations of Vibrio parahaemolyticus to understand their distribution, form and type and to develop a novel method.for the detection of Vibrio parahaemolyticus. At present,it is reported that Vibrio parahaemolyticus can be detected by loop mediated isothermal amplification (LAMP). However, these are still some problems, such as costly, time-consuming, instability, and easy contamination.So, a novel method for the detection of Vibrio parahaemolyticus is urgently needed.Objective To understand the situation of the commercial seafood contaminated by Vibrio parahaemolyticus and to establish simple and convenient, rapid and sensitive method for the detection of genes (tlh, tdh, trh) in Vibrio parahaemolyticus by LAMP.Methods Part 1: Total of 5 categories, 20 konds and 161 samples from fish, shrimp, shellfish, crabs and cephalopods was collected in the season of highly food poisoning. And then Vibrio parahaemolyticus were isolated and identified by traditional culture method and physiological and biochemical method, respectly. Then, the gene tlh was verified by PCR. Then, the toxicity genes (tdh and trh) were detected by PCR and their distribution was known through statistical analysis.At last, the research on the serum cluster of the isolated and verified Vibrio parahaemolyticus was carried out by 11 kinds of O group serum for Clustering. Part 2: The species-specific gene, thermolabile hemolysin (tlh) of Vibrio parahaemolyticus was selected to design 1 set of LAMP primers across 6 distinct regions. Set up the LAMP method for detecting Vibrio parahaemolyticus by optimizing the condition of reaction including the concentration of Mg2+ and betaine, FIP/BIP ratio, LF or not, and the condition of reaction temperature and reaction time. Then, 74 strains of experimental bacterial were detected by LAMP to evaluate its specificity and gradient dilution of standard bacterial genetic DNA, pure culture of Vibrio parahaemolyticus and shellfish samples were detected by LAMP to evaluate its sensitivity. The applied value of this method was estimated through the comparison of the detection of the same 74 samples by general PCR. Part 3: The species-specific genes, hemolytic toxin genes of heat direct hemolysin gene;heat-related hemolysin hemolysin gene (tdh and trh) in Vibrio parahaemolyticus were selected to design 1 set of LAMP primers through 6 distinct regions respectively. Then, 74 strains of foodborne pathogens were selected to dectect the tlh and tdh gene by the novel LAMP method. for their specificity and on-site application value.Results:Part 1: 45 strains of Vibrio parahaemolyticus from 161 samples were isolated and verified by traditional methods and PCR, in which 7 strains contain tdh gene and 0 strain contains trh gene. The average of relevance ratio is 27.95% and there is a significant difference among different categories of aquatic product (χ2 = 11.20, 0.01 <p <0.05). 45 strains of Vibrio parahaemolyticus were divided into 7 serotypes, O:1 (1 / 45, 2.2 %), O:3 (11/45, 24.4%), O:4 (7 / 45, 15.6%), O:5 (9 / 45, 20.0%), O:9 (2 / 45, 4.4%), O: 10 (4 / 45, 8.9%), O:11 (12/45, 26.7%). Part 2: The optimization showed that at the conditions of 8 mM Mg2+, 0M betaine, 8:1 ratio of BIP/FIP, 40mM LF, 60℃and 55 minutes, the amplification can carry out successfully. The detection of all the Vibrio parahaemolyticus in 74 experimental strains were positive, while the control samples were negative.The minimum limits of standard bacterial genetic DNA, pure culture of Vibrio parahaemolyticus and shellfish samples were 1fg, 2.53×102CFU/mL (1000 times than PCR), 9.42×102CFU/g (1000 times than PCR), respectively. Part 3: 7 strains of Vp in 74 strains of foodborne pathogens were detected to be tdh gene positive, while 1 strain of Vp in 74 strains of foodborne pathogens were detected to be trh gene positive.Conclusion:This study provided some reference to the distribution of Vibrio parahaemolyticus in the commercial aquatic products through the routine investigation. The method of LAMP for detecting tlh, tdh and trh in Vibrio parahaemolyticus is simple and convenient, rapid, highly sensitive and highly specific and is a potential method for one-site detection of Vibrio parahaemolyticus after further optimization, perfection and promoting.